The present invention relates generally to photothermal imaging tiny particles immersed in a given medium. More particularly the present invention relates to photothermal detection of metallic particles that can serve as a label for organelles or organic compounds and biomolecules included in a living cell.
Ambient optical detection of labeled molecules is presently limited to fluorescent dyes by photobleaching and semiconducting particles of nanometer size by blinking effects.
Nanometer-sized metal particles do not optically bleach and appear therefore particularly useful to serve as optical labels, provided suitable detection process can be found.
An ideal optical label for large molecules should generate an intense optical signal, be of very small size, prove durable in time, as well as chemically inert and easy to bind to the molecule of interest in a controlled manner.
All present-day known optical markers fall short of the ideal label status definition.
The most common known labels, like fluorescent dyes, can usually be grafted to the molecule of interest. Their red-shifted fluorescence can be sifted very efficiently out of the given background.
The main drawback they are known to suffer however is photobleaching, i.e. an irreversible photochemical process leading from the excited fluorophore status to a non-fluorescent product.
Nanocrystals of II-VI semi-conductors, such as CdSe/ZnS have recently been proposed as optical markers. See particularly M. Bruchez, Jr., M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, in Science 281, 2013-6 1998 and W. C. Chan, S. Nie, in Science 281, 2016-8, 1998.
Although the semi-conductors resist bleaching longer than dyes, their luminescence brightness is liable to blinking while they are difficult to functionalize in a controlled way.
In contradistincton, metal particles are known to be currently used for single-particle or single-molecule tracking and immocytochemistry, see as an example W. Baschong, J. M. Lucocq and J. Roth, Histochemistry 83, 409-11 (1985) or J. W. Slot and H. J. Geuze, Eur J. Cell Biol 38, 87-93 (1985).
The above mentioned metal particles can either take the form of colloids with diameters ranging between a micron and a few nanometers or synthesized clusters with well-defined chemical structures. See for example P. A. Frey and T. G. Frey. J Struct Biol 127, 94-100 (1999) or J. F. Hainfeld and R. D. Powell J Histochemistry Cytochemistry 48, 471-80 (2000).
Sub-micrometer metal particles down to diameters of 40 nm can be imaged using an optical microscope by means of their Rayleigh given, by illuminating in dark field at the plasmon frequency, with differential interference contrast (DIC) and video enhancement, or with total reflexion. See particularly S. Schultz, D. R. Smith, J. J. Mock and D. A. Schultz in Proc. Natl. Acad. Sci. USA 97, 996-1001 (2000), J. Gelles, B. J. Schnapp, M. P. Sheetz, in Nature 331, 450-3 (1988) and C. Sxc3x6nnischen et al in Applied Physics Letters 77, 2949-2951 (2000) respectively for the three preceding mentioned alternatives.
While metal particles are very appealing optical labels owing to their absence of photobleaching phenomenon and optical saturation at reasonable exciting intensities, the Rayleigh given phenomenon they undergo decreases like the sixth power of their diameter, with the given signal being to be discriminated from a strong background.
Therefore the minimum size in diameter of a particle being detected in a living cell or in a given tissue is in practice well above the theoretical limit of 40 nm in diameter.
The well known Electron microscopy with its superior spatial resolution can well distinguish particles with diameters as low as 5 nm from organelles in a cell. See particularly J. M. Robinson, T. Takizawa, D. D. Vandre in J. Microscience 199, 163-79 (2000). Unfortunately Electron microscopy cannot be operative at ambient conditions.
The present invention provides for a method and device for photothermal imaging tiny metal particles immersed in a given medium particularly adapted to remedy the drawbacks suffered by the methods, processes and devices of the prior art.
The method and the device which are the object of the present invention distinguish over the method and device of the copending provisional application U.S. Ser. No. 60/410,305 filed on Sep. 13, 2002, now filed as the complete specification U.S. Ser. No. 10/386,937 filed on Mar. 13, 2003 in that detecting the photothermal effect and corresponding induced phase changes takes place now with highly reduced illuminating power, without inducing any damage or detrimental effect to living bodies like cells whose organelles or tiny compounds elements can thus be submitted to fine labeling.
More particularly one object of the present invention is to provide for a method and device for photothermal imaging small particles, metal particles, down to 1 nm in diameter at ambient conditions with an optical microscope.
Another object of the present invention is thus to provide for a method and device for photothermal imaging small particles allowing thus to correlate single particle, as labels, with optical microscopic images, without any need for conjugation to bulky fluorescent antibodies.
Another object of the present invention is thus to provide for a very high sensitive method and device particularly adapted to allow an efficient, reproductible and promising way to visualize low amounts of proteins biomolecules or organelles in living cells or belonging to the membrane thereof.
According to the method for photothermal imaging tiny metal particles immersed in a given medium which is the object of the invention, the given medium is deposited on a transparent glass slide.
The given medium and immersed tiny particles are illuminated through separate phase reference laser beam and sensitive probe laser beam the sensitive probe laser beam undergoing through impingement on the given medium slight phase changes induced by photothermal effect due to a local heating within the given medium, in the absence of any substantial phase changes to the phase reference laser beam.
Illuminating is performed by focusing the separate phase reference laser beam and sensitive probe laser beam through the transparent glass slide at a given depth within the given medium from the output face of the transparent glass slide, transmitted phase reference laser beam and transmitted sensitive probe laser mean undergoing these slight phase changes induced by photothermal effect due to a local heating being thus generated.
An image of the given medium and tiny particles at the given depth through the transmitted phase reference laser beam and transmitted sensitive probe laser beam is formed.
The slight phase changes on the transmitted sensitive probe laser beam with reference to the transmitted phase reference laser beam are detected on the image through differential phase interference contrast phenomenon, with each of the tiny metal particles immersed in the given medium being imaged as an optical label.
The device for photothermal imaging of tiny metal particle immersed in a given medium which is the object of the invention comprises a unit for illuminating part of this given medium and immersed tiny particles through separate phase reference laser beam and sensitive probe laser beam, the transmitted sensitive probe laser beam undergoing through impingement of the sensitive probe laser beam on the given medium slight phase changes induced by photothermal effect due to a local heating within the given medium, in the absence of any substantial phase changes to the transmitted phase reference laser beam.
It further comprises a unit for detecting these slight phase changes on the transmitted sensitive probe laser beam with reference to the transmitted phase reference laser beam through a differential phase interference contrast phenomenon. A unit is provided for imaging each of the tiny metal particles immersed within the given medium as an optical label from the differential phase interference contrast phenomenon.
The objectives, advantages and particulars of the present invention will be understood by reading the following detailed description and accompanying drawings.